ANTICANCER RESEARCH 35: 4625-4632 (2015)

WNT16-expressing Acute Lymphoblastic Leukemia Cells are Sensitive to Autophagy Inhibitors after ER Stress Induction

MELETIOS VERRAS1, IOANNA PAPANDREOU2 and NICHOLAS C. DENKO2

1General Biology Laboratory, School of Medicine, University of Patra, Rio, Greece; 2Department of Radiation Oncology, Wexner Medical Center and Comprehensive Cancer Center, The Ohio State University, Columbus OH, U.S.A.

Abstract. Background: Previous work from our group showed burden of proteins in the ER through decreased translation, hypoxia can induce endoplasmic reticulum (ER) stress and increased chaperone expression, and increased removal of the block the processing of the WNT3 protein in cells engineered malfolded proteins through degradation. If the cell is unable to express WNT3a. Acute lymphoblastic leukemia (ALL) cells to relieve the ER stress, then cellular death can ensue (3). with the t(1:19) translocation express the WNT16 gene, which The microenvironment of solid tumors is often poorly is thought to contribute to transformation. Results: ER-stress perfused, resulting in regions of hypoxia and nutrient blocks processing of endogenous WNT16 protein in RCH-ACV deprivation (4, 5). However, hypoxia has been also shown to and 697 ALL cells. Biochemical analysis showed an impact cancer of the bone marrow such as aggressive aggregation of WNT16 proteins in the ER of stressed cells. leukemia (6). In addition to inducing the hypoxia-inducible These large protein masses cannot be completely cleared by factor 1 (HIF1) transcription factor, severe hypoxia induces ER-associated protein degradation, and require for additional stress in the ER (7, 8). Cells with compromised ability to autophagic responses. Pharmacological block of autophagy respond to ER stress (due to genetic ablation of the x-box significantly increased cell death in ER-stressed ALL. binding protein (XBP1) transcription factor or the Protein Furthermore, murine cells engineered to express WNT16 are kinase RNA- like endoplasmic reticulum kinase (PERK)) similarly sensitized. Conclusion: ALL cells expressing WNT16 grow poorly as model tumors (9, 10). Pharmacological are sensitive to ER stress, and show enhanced killing after inhibition of ER stress responses can also have anticancer addition of chloroquine. These findings suggest a potential effects in vitro and in model myeloma (11). Hypoxia-induced clinical application of inducers of ER stress with inhibitors of ER stress can inhibit the secretion and paracrine activity of autophagy in patients with high-risk ALL. growth factors such as WNT family members (12). WNT proteins have highly conserved series of 25-27 cysteine Environmental conditions or drugs can interfere with the residues that are thought to establish a complex tertiary proper functioning of the endoplasmic reticulum (ER) (1). A structure essential for their activity (13). Lack of oxygen block to the normal flow of proteins through the ER results in disrupts the normal formation of disulphide bonds in the ER stress and elicits the compensatory unfolded protein WNT proteins, leading to their retention in the ER and response (UPR) (2). Proteins can accumulate in the ER in an ultimately in their degradation through proteasomal and improperly folded state after a block of essential ER functions autophagic mechanisms (12). Activation of autophagy under such as protein folding, disulfide bond formation, or ER stress conditions is therefore compensatory and leads to glycosylation. Due to the presence of unfolded proteins in the the degradation of unfolded/misfolded protein aggregates ER, cells initiate a series of responses designed to reduce the that are not soluble and cannot be degraded by ER associated degradation (ERAD) (14, 15). We have previously shown that hypoxia induces autophagy via AMP activated protein kinase (AMPK) Correspondence to: Nicholas C. Denko, Department of Radiation activity, in an HIF-independent process (16). We have also Oncology, Wexner Medical Center and Comprehensive Cancer shown that hypoxic ER stress can inhibit the processing of Center, Ohio State University, Columbus OH 43210, U.S.A. E-mail: the WNT family of secreted glycoproteins in engineered [email protected] cancer cells (12). In the present study, we again use WNT Key Words: Unfolded protein response, tumor hypoxia, acute glycoproteins as tools to explore the hypothesis that lymphoblastic leukemia, WNT growth factors, ER-associated autophagy is integral to the hypoxia-induced ER stress degradation. response. Here we report studies examining expression of

0250-7005/2015 $2.00+.40 4625 ANTICANCER RESEARCH 35: 4625-4632 (2015) endogenous WNT16 protein in pre-B acute lymphoblastic Results leukemia (ALL) cell lines after treatment with conditions that induce ER stress. Expression of WNT16 is compromised by ER stress in ALL cells. Previous work from our group examined the processing Materials and Methods of WNT3A in cells engineered to express the protein after ER stress. We engineered murine L cells and human RKO colorectal cancer cells to express exogenous WNT3A and Cells, cell culture and reagents. ProB leukemic cell lines RCH-ACV and 697 cells were cultured in RPMI with 20% fetal bovine serum found that WNT processing was impaired during ER stress (FBS), Murine fibroblast L cells were cultured in Dulbecco’s from hypoxia, and this down-regulated β-catenin signaling. modified eagle medium (DMEM) with 10% FBS. For moderate We now wished to extend these studies to determine if tumor hypoxia, cells were treated in a variable-oxygen Invivo2 humidified cells that naturally expressed WNT family members were hypoxia workstation (Ruskinn Technologies, Bridgend, UK). Severe also sensitive to ER stress. Pre-B ALL cells that contain the hypoxia was generated in an anaerobic workstation gassed with 5% fusion protein which joins the transactivation domain of the CO , 5% H and 95% N containing a palladium catalyst (Sheldon 2 2 2 E2A protein with the DNA binding domain of the Co., Cornelius, OR, USA). Transient and stable transfections were performed using Lipofectamine (Invitrogen, Carlsbad, CA, USA). homeoprotein PBX express WNT16 due to the activity of the MG-132, tunicamycin, thapsigargin, chloroquine, E64 and pepstatin fusion protein (17, 18). We therefore obtained RCH-ACV were purchased from Sigma-Aldrich (Saint Louis, MO, USA). and 697 ALL cells and examined WNT16 expression and Dithiothreitol (DTT) was purchased from Invitrogen. The pLPC- biological activity by western blot after treatment with ER Wnt16 and pLPC empty retroviral vectors were a kind gift from Dr. stressing agents. Figure 1 shows that treatment with reduced Amato Giaccia. oxygen tension causes reduced expression of endogenous WNT16 in both cell lines (Figure 1A and B). Furthermore, Retroviral transduction. WNT16 expressing cells were generated by retroviral transduction. A WNT16-expressing retroviral vector we also show that there is decreased WNT signaling because (pLPC-WNT16) was transfected into HEK 293 Phoenix cells using there is less stabilized β-catenin in these cells after treatment Lipofectamine as directed by the manufacturer (Life Technologies, with hypoxia (Figure 1A and B). Grand Island NY USA). After 48 h, the supernatant containing the We confirmed that the decreased WNT16 protein is due to retrovirus was collected, filtered and used to transduce the indicated incomplete processing in the ER using the thiols protection cell lines in the presence of 5 μg/ml polybrene (Sigma Aldrich, St assay. Extracts were collected in N-ethylmaleimide and this Louis MO, USA). WNT16-positive clones were selected using stabilized the thiols and disulphide bonds in the WNT16 puromycin, and expression confirmed by immunoblot. protein. The samples were electrophoresed either on a non- Western blotting. In brief, treated cells were harvested in RIPA buffer, reducing (Figure 1C left) or reducing (Figure 1C right) SDS lysates were sonicated, cleared by centrifugation, and protein polyacrylamide gel. Figure 1C shows that without addition concentrations were quantitated by BCA reagent (Life Technologies, of DTT to break disulphide bonds, a high fraction of the Grand Island NY USA). Proteins (25–50 μg) were electrophoresed on WNT16 proteins extracted from hypoxia-treated cells a reducing Tris-Tricine gel, and electroblotted to polyvinyl difluoride migrate as very large protein aggregates with apparent membrane. Antibodies used were mouse anti-β-catenin (BD velocity indicating sizes above 250 kDa. Addition of DTT to Biosciences Pharmingen San Diego CA USA), mouse anti-human WNT16 (BD Biosciences Pharmingen), LC3 (MBL International these extracts breaks all disulfide bonds and collapses the Woburn MA USA), and mouse anti-β-actin (Abcam Hong Kong). WNT16 reactive material to an apparent monomeric size of Primary antibodies were detected with species-specific horseradish 45 kDa. We interpret these results to indicate that severe peroxidase secondary antibodies (DAKO Carpenteria CA USA) and hypoxia interferes with correct disulphide bond formation of visualized with ECL (Amersham Piscataway NJ USA) on a Storm 860 WNT16 proteins in the ER, because oxygen is the terminal phosphoimager (Molecular Devices San Francisco CA USA). electron acceptor in the reaction forming disulphide bonds Thiol modification blocking assay (treatment with N-ethylmaleimide). (7). Without correct 3-dimensional structure, WNT proteins Cells were cultured for 24 h and then lysed in RIPA buffer (1% aggregate, and this signals for their destruction. We also Triton X-100, 150 mM NaCl, 20 mM Hepes (pH 7.5), 10% glycerol, directly tested cellular processing of WNT16 to classical ER 1 mM EDTA, 100 mM NaF, 17.5 mM β-glycerophosphate, 1 mM stressor tunicamycin which inhibits N-linked glycosylation phenylmethylsulfonyl fluoride, 4 μg/ml aprotinin, 2 μg/ml pepstatin and DTT which inhibits disulphide bond formation in the A) supplemented with fresh 20 mM N-ethylmaleimide (NEM; cell. Western blot analysis of lysates from these cells shows Sigma-Aldrich). Cell lysates (30 μg) were analyzed on an 8% that both these stresses had a profound effect, and reduced sodium dodecyl sulfate-polyacrylamide gel under reducing (125 mM WNT16 protein expression in RCH-ACV cells (Figure 1D). DTT) and non-reducing (0 mM DTT) conditions.

Statistical analysis. Pairwise comparisons were made by two-tailed Reduced WNT16 expression after ER stress is due to Students t-test on primary data points. Statistical significance was proteolysis. We have shown that severe hypoxia can accepted at p<0.05. stimulate autophagy in carcinoma cells (16). We, therefore,

4626 Verras et al: ER Stress Sensitizes WNT16-expressing Leukemic Cells

Figure 1. ER stress causes WNT16 aggregation and destruction. A: 697 Leukemic cells expressing WNT16 were treated for 24 h with increasingly severe levels of hypoxia as indicated, and WNT16 protein levels were then detected by western blot. WNT16 activity was determined by detection of the level of stabilized β-catenin. Note that as WNT levels decrease, β-catenin levels decrease accordingly. B: RCH-ACV leukemic cells were treated similarly and WNT16 levels and activity detected as in (A). C: Determination of WNT protein aggregates in cells treated with severe hypoxia by means of the thiol protection assay and migration through non-reducing and reducing polyacrylamide gels. Under non-reducing conditions, a large fraction of WNT16 from hypoxia-treated cells migrated as a slow aggregate, with apparent mass well above 200 kDa. When these samples were reduced and disulphide bonds broken, monomeric WNT16 was detected. Comparison of left and right panels indicates WNT16 aggregates were due to aberrant disulphide bond formation. D: WNT16 protein expression is also sensitive to classical drug-induced ER stresses. WNT levels were determined after treatment with tunicamycin (to inhibit glycosylation) or DTT (to directly inhibit disulphide bond formation).

examined WNT16-expressing leukemia cells for markers of hypoxia, both inhibitors resulted in a partial restoration of autophagy after treatment with ER stress. Figure 2A shows WNT protein levels. These results suggest that both proteasome that 697 cells treated with either chemical ER stress or and autophagasome/cathepsins contribute to reduced WNT16 severe hypoxia displayed an increased processing of myosin- protein expression after ER stress from hypoxia. associated light chain protein 3 (LC3-II) that is a marker of active autophagy (19). Similarly, there was increased ER stress-induced toxicity can be potentiated in ALL cells turnover of the adaptor protein p62/sequestrosome 1 in 697 with autophagy inhibitors. We next treated ALL cells with and RCH-ACV cells treated with hypoxia, indicating either ER stress-inducing agents, autophagy inhibitors, or autophagic turnover of protein aggregates (20). both, and determined the dead fraction by trypan blue In order to determine if the reduction of WNT16 protein was exclusion. Figure 3A shows that when RCH-ACV cells are due to proteolysis by the proteasome or the autophagasome, we treated with hypoxia there was very modest toxicity. used inhibitors of each of these processes in ER-stressed cells. Similarly, the autophagy inhibitors E64/pepstatin and Figures 2B and C show that under normoxia, neither inhibitor chloroquine were not very toxic to normoxic cells. However, had an effect on WNT16 expression level. However, in when the autophagy inhibitors were combined with hypoxia,

4627 ANTICANCER RESEARCH 35: 4625-4632 (2015)

Figure 2. Turnover of WNT16 after ER stress is partly through autophagy A: Analysis of myosin associated light chain 3 processing in 697 cells exposed to chemical ER stress or severe hypoxia for 12 or 24 h (top panel). Increased LC3-II indicates processing of LC3 at the autophagolysosome. Decrease of adaptor protein p62 was noted in both 697 cells (middle panel) and RCH-ACV cells (bottom panel) exposed to hypoxia. Note that a decrease in p62 indicates turnover of protein aggregates in the autophagolysosome. B: Partial rescue of WNT16 protein levels in RCH-ACV cells exposed to severe hypoxia for 24 h by addition of inhibitors of the proteasome, or the lysosomal proteases for the final 2 h. Note that treatment of normoxic cells with protease inhibitors did not alter WNT16 level. C: Partial rescue of WNT16 protein expression in ER-stressed 697 cells treated similarly to the RCH-ACV cells shown in (B).

Figure 3. Toxicity due to ER stress can be potentiated by the addition of autophagy inhibitors. A: Killing of RCH-ACV cells exposed to 72 h of severe hypoxia or hypoxia with the autophagy inhibitors . There was very modest toxicity in the cells treated with hypoxia alone, but addition of the autophagy inhibitors greatly enhanced cell killing as measured by trypan blue exclusion. N=3 independent experiments performed in triplicate. B: Killing of RCH-ACV cells by exposure to 24 h of the chemical ER stresses tunicamycin, Thapsigargin or DTT as indicated, and the potentiation of the killing from the ER stress by the addition of chloroquine. N=3 Independent experiments performed in triplicate. Statistical significance: *0.05>p>0.01, **0.01>p>0.001, and ***p<0.001.

4628 Verras et al: ER Stress Sensitizes WNT16-expressing Leukemic Cells

Figure 4. Sensitization of mouse L cells to ER stress and autophagy inhibitors by the introduction of WNT16 gene. A: Western blot of lysates from L cells transduced with empty vector, or those transduced with retrovirus expressing WNT16 as indicated. B: Reduction of WNT16 protein expression in L cells exposed to 24 h of severe hypoxia and rescue of the expression by addition of either MG132 to inhibit the proteasome, or chloroquine to inhibit the lysosomal cathepsins for the final 2 h. C: L cells expressing WNT16 are specifically sensitized to killing by the combination of DTT to induce ER stress and chloroquine to block autophagy. Cells were treated for 72 h and the dead fraction determined by trypan blue exclusion. N=2 independent experiments performed in triplicate. D: ER-stress dependent killing of L cells expressing WNT16 was also measured by colony-formation assay after plating of 300 cells. This assay also shows specific killing of the WNT16-expressing variant line. N=2 independent experiments performed in triplicate. Statistical significance: *0.05>p>0.01, and **0.01>p>0.001.

there was significant cellular death (Figure 3A). These expressed WNT16 in murine L cells that are commonly used experiments were repeated with chemical inducers of ER to produce WNT proteins in a cell that does not rely on the stress and resulted in qualitatively similar results, although biological effects of the growth factor for survival (21). the drugs themselves did have some toxicity such that the Figure 4A shows the stable expression of the engineered increased toxicity with autophagy inhibitors led to a smaller WNT16 in L cells after transduction with recombinant fold increase (Figure 3B). Thapsigargin and DTT led to retrovirus. Figure 4B shows that the expression of WNT16 significant increase in cell death with the addition of in these cells is significantly reduced after treatment with chloroquine, while tunicamycin tended to increase toxicity hypoxia, and this reduction can be reversed with treatment (p=0.06). of chloroquine or E64/pepstatin. Finally, Figure 4C and D show that cells expressing WNT16 protein are specifically Expression of WNT16 in mouse L cells sensitizes them to ER sensitized to the disulphide bond disrupter DTT when it was stress in combination with autophagy inhibitors. In order to combined with the autophagy inhibitor chloroquine. Figure molecularly connect WNT16 expression with cellular 4C shows the acute toxicity after 72 h treatment, and Figure sensitivity to the two-drug cocktail, we needed a genetically- 4D shows the colony formation after 9 days growth of the matched system that only differed by the expression of the cells in the indicated media. Both measures indicate that one gene. However, the ALL cells are dependent on WNT there was significant death only of the WNT16-expressing signaling for survival (18), hence it is not possible to cells, and only in the combination treatment with ER stress knockdown WNT16 in these cells. As an alternative, we and chloroquine.

4629 ANTICANCER RESEARCH 35: 4625-4632 (2015)

Discussion or conditions that cause ER stress. This sensitivity can be further exploited in a fraction of acute leukemia by the These results support our previous findings and the concept addition of inhibitors of autophagy. Identifying cancer types that hypoxia is a negative regulator of WNT processing and that express WNT proteins (especially those addicted to WNT signaling because it blocks WNT protein in the ER, and protein signaling) may identify those that would be sensitive leads to its ultimate destruction. The WNT protein that to such a therapeutic approach. accumulates in the ER has improper disulfide bonds and generates toxic protein aggregates. These aggregates must be Acknowledgements cleared by an autophagic process because the insoluble aggregates are resistant to ERAD. The Authors would like to thank Drs Roel Nusse and Michael Accumulation of malfolded proteins in the ER elicits the Cleary for cell lines, Dr Amato Giaccia for his gift of the pLC cell to respond with the UPR and the activation of the three constructs, and Dr Albert Koong for his helpful discussions. arms of the UPR: PERK-dependent induction of activating transcription factor 4 (ATF4), endoplasmic reticulum to References nucleus signaling 1 (IRE1/ERN1)-dependent XBP1 splicing and proteolytic activation of ATF6. Together these three arms 1 Sovolyova N, Healy S, Samali A and Logue SE: Stressed to of the response attempt to decompress the ER by slowing the death - mechanisms of ER stress-induced cell death. Biol Chem introduction of new proteins into the ER, and enhancing 395: 1-13, 2014. proper folding of those proteins already in the ER through 2 Merksamer PI and Papa FR: The UPR and cell fate at a glance. J Cell Sci 123: 1003-1006, 2010. increased chaperone expression, and removal of the proteins 3 Sano R and Reed JC: ER stress-induced cell death mechanisms. deemed unrepairable. It is well established that the ERAD Biochim Biophys Acta 1833: 3460-3470, 2013. pathway is an efficient means of removal of proteins that can 4 Ackerman D and Simon MC: Hypoxia, lipids, and cancer: be transported across the ER membrane and into the surviving the harsh tumor microenvironment. Trends in cell cytoplasm for proteasomal destruction (22). However, if biology 24: 472-478, 2014. these proteins become aggregated into insoluble tangles, then 5 Denko NC: Hypoxia, HIF1 and glucose metabolism in the solid the ERAD pathway is not efficient, and an autophagic tumour. Nat Rev Cancer 8: 705-713, 2008. process must compensate for their removal (23). 6 Benito J, Zeng Z, Konopleva M and Wilson WR: Targeting hypoxia in the leukemia microenvironment. Int J Hematol Oncol Paracrine and autocrine WNT signaling has been suggested 2: 279-288, 2013. as a mechanism for maintaining 'stemness' and aggressiveness 7 Feldman DE, Chauhan V and Koong AC: The unfolded protein of cancer cells (24, 25). However, these findings suggest that response: a novel component of the hypoxic stress response in it may not be quite as simple in vivo for cells in hypoxic tumors. Mol Cancer Res 3: 597-605, 2005. regions of tumors or bone marrow to produce and secrete 8 Koumenis C, Bi M, Ye J, Feldman D and Koong AC: Hypoxia WNT growth factors to simulate canonical and non-canonical and the unfolded protein response. Methods in enzymology 435: β-catenin signaling. Furthermore, WNT gene expression may 275-293, 2007. 9 Blais JD, Addison CL, Edge R, Falls T, Zhao H, Wary K, even be detrimental to the growth of tumor cells growing in Koumenis C, Harding HP, Ron D, Holcik M and Bell JC: Perk- microenvironmental hypoxia, as compared to activation of β- dependent translational regulation promotes tumor cell catenin by mutation (12). adaptation and angiogenesis in response to hypoxic stress. Mol The ability of cells to adapt to environmental stress appears Cell Biol 26: 9517-9532, 2006. to be essential to the growth of cells in solid tumors (9, 10). 10 Romero-Ramirez L, Cao H, Nelson D, Hammond E, Lee AH, However, hypoxia exists in the bone marrow in health and Yoshida H, Mori K, Glimcher LH, Denko NC, Giaccia AJ, Le disease and influences leukemic progression (26-28). These QT and Koong AC: XBP1 is essential for survival under hypoxic conditions and is required for tumor growth. Cancer Res 64: studies indicate that hypoxia is an area for additional study to 5943-5947, 2004. establish its importance in the natural history of diseases of the 11 Papandreou I, Denko NC, Olson M, Van Melckebeke H, Lust S, bone marrow. These findings also support the concept that Tam A, Solow-Cordero DE, Bouley DM, Offner F, Niwa M and rational design of combination chemotherapy may include Koong AC: Identification of an Ire1alpha endonuclease specific drugs that influence ER stress (29). If microenvironmental inhibitor with cytotoxic activity against human multiple hypoxia can initiate a condition of ER stress, perhaps addition myeloma. Blood 117: 1311-1314, 2011. of chemotherapeutic agents that enhance ER stress, such as 12 Verras M, Papandreou I, Lim AL and Denko NC: Tumor hypoxia bortezomib, could be combined with anti-autophagy agents, blocks Wnt processing and secretion through the induction of endoplasmic reticulum stress. Molecular and cellular biology 28: such as chloroquine, in order to achieve therapeutic gain. 7212-7224, 2008. WNT proteins have a complex three-dimensional structure. 13 Cho SJ, Valles Y, Giani VC Jr., Seaver EC, and Weisblat DA: Expression of WNT proteins in cancer cells can promote Evolutionary dynamics of the wnt gene family: a lophotrochozoan aggressive cancer growth but can also sensitize them to agents perspective. Mol Biol Evol 27: 1645-1658, 2010.

4630 Verras et al: ER Stress Sensitizes WNT16-expressing Leukemic Cells

14 Ding WX, Ni HM, Gao W, Hou YF, Melan MA, Chen X, Stolz 24 Scheel C, Eaton EN, Li SH, Chaffer CL, Reinhardt F, Kah KJ, DB, Shao ZM, and Yin XM: Differential effects of endoplasmic Bell G, Guo W, Rubin J, Richardson AL and Weinberg RA: reticulum stress-induced autophagy on cell survival. J Biol Chem Paracrine and autocrine signals induce and maintain 282: 4702-4710, 2007. mesenchymal and stem cell states in the breast. Cell 145: 926- 15 Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, 940, 2011. Murakami T, Taniguchi M, Tanii I, Yoshinaga K, Shiosaka S, 25 ten Berge D, Kurek D, Blauwkamp T, Koole W, Maas A, Eroglu Hammarback JA, Urano F, and Imaizumi K: Autophagy is E, Siu RK and Nusse R: Embryonic stem cells require Wnt activated for cell survival after endoplasmic reticulum stress. proteins to prevent differentiation to epiblast stem cells. Nat Cell Molecular and cellular biology 26: 9220-9231, 2006. Biol 13: 1070-1075, 2011. 16 Papandreou I, Lim AL, Laderoute K, and Denko NC: Hypoxia 26 Ng KP, Manjeri A, Lee KL, Huang W, Tan SY, Chuah CT, signals autophagy in tumor cells via AMPK activity, independent Poellinger L and Ong ST: Physiologic hypoxia promotes of HIF-1, BNIP3, and BNIP3L. Cell Death Differ 15: 1572- maintenance of CML stem cells despite effective BCR-ABL1 1581, 2008. inhibition. Blood 123: 3316-3326, 2014. 17 Casagrande G, te Kronnie G and Basso G: The effects of siRNA- 27 Silveira VS, Freire BM, Borges KS, Andrade AF, Cruzeiro GA, mediated inhibition of E2A-PBX1 on EB-1 and Wnt16b Sabino JP, Glass ML, Yunes JA, Brandalise SR, Tone LG and expression in the 697 pre-B leukemia cell line. Haematologica Scrideli CA: Hypoxia-related gene expression profile in 91: 765-771, 2006. childhood acute lymphoblastic leukemia: prognostic 18 Mazieres J, You L, He B, Xu Z, Lee AY, Mikami I, McCormick implications. Leuk Lymphoma 55: 1751-1757, 2014. F and Jablons DM: Inhibition of Wnt16 in human acute 28 Velasco-Hernandez T, Hyrenius-Wittsten A, Rehn M, Bryder D lymphoblastoid leukemia cells containing the t(1;19) and Cammenga J: HIF-1alpha can act as a tumor suppressor translocation induces apoptosis. Oncogene 24: 5396-5400, 2005. gene in murine acute myeloid leukemia. Blood 124: 3597-3607, 19 Mizushima N and Yoshimori T: How to interpret LC3 2014. immunoblotting. Autophagy 3: 542-545, 2007. 29 Kharabi Masouleh B, Geng H, Hurtz C, Chan LN, Logan AC, 20 Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen Chang MS, Huang C, Swaminathan S, Sun H, Paietta E, Melnick H, Overvatn A, Bjorkoy G and Johansen T: p62/SQSTM1 binds AM, Koeffler P and Muschen M: Mechanistic rationale for directly to Atg8/LC3 to facilitate degradation of ubiquitinated targeting the unfolded protein response in pre-B acute protein aggregates by autophagy. J Biol Chem 282: 24131- lymphoblastic leukemia. Proc Natl Acad Sci USA 111: E2219- 24145, 2007. 2228, 2014. 21 Shibamoto S, Higano K, Takada R, Ito F, Takeichi M and Takada S: Cytoskeletal reorganization by soluble Wnt-3a protein signalling. Genes Cells 3: 659-670, 1998. 22 Merulla J, Fasana E, Solda T and Molinari M: Specificity and regulation of the endoplasmic reticulum-associated degradation machinery. Traffic 14: 767-777, 2013. 23 Benbrook DM and Long A: Integration of autophagy, Received May 7, 2015 proteasomal degradation, unfolded protein response and Revised June 10, 2015 apoptosis. Exp Oncol 34: 286-297, 2012. Accepted June 12, 2015

4631